Comparative Analysis of Chemical Composition andAntibacterial and Anti-Inflammatory Activities of theEssential Oils from Chrysanthemum morifolium ofDifferent Flowering Stages and Different Parts

The inflorescence of Chrysanthemum morifolium Ramat., a well-known traditional Chinese herb, has been proved to have a certain inhibitory effect on some bacteria; however, its main components and acne bacteria inhibition effect remain to be elucidated. In this study, GC-MS was used to analyze the components of different flowering stages and different parts and to study the inhibitory effects of six essential oils on S. aureus and P. acnes and their alleviating effects on THP-1 cell inflammation. GC-MS combined with relative retention index method analyzed results stated that the 5 samples of C. morifolium to detect the 124 kinds of volatile components, including (E)-tibetin spiroether, are first detected in the volatile oil of the C. morifolium, and the content of (E)-tibetin spiroether is higher in immature inflorescence of C. morifolium and decreases as it extends its flowering period. Furthermore, the research results of inhibiting common acne-causing bacteria showed that the bacteriostatic effect of essential oils from JH at different flowering stages was better than that from JM and TJ, while the bacteriostatic effect of essential oil from stem and leaf of C. morifolium (SLC) at different parts was better than the roots of C. morifolium (RC). Finally, it was proved that the essential oil from SLC and C. morifolium could alleviate the inflammation of THP-1 cells induced by P. acnes. In conclusion, the antibacterial and anti-inflammatory effects of C. morifolium essential oil may be related to heterospiroolefins compounds, and the antibacterial activity decreases with the prolongation of flowering stage. It was suggested that volatile oil from C. morifolium and SLC could be used as effective components of antibacterial and anti-inflammatory cosmetics.


Introduction
e Chrysanthemum genus (Chrysanthemum morifolium Ramat.) belongs to the Compositae family, mainly distributed in temperate regions, and has been widely used as medicinal, edible, and ornamental plants for thousands of years [1,2]. ere are more than 200 species and varieties of Compositae plants, including Chrysanthemum indicum, Chrysanthemum trifurcatum (Desf.), and Chrysanthemum japonense, all of which have remarkable pharmacological activity [3][4][5]. e capitulum of Chrysanthemum morifolium Ramat. was used as Hangbaiju in China and is rich in volatile oils, flavonoids, phenolic acids, and polysaccharides. Recently, pharmacological studies have revealed that it has broad bioactivities, such as anti-inflammatory, antitumor, relieving diabetes, and antioxidative damage activities [6,7]. It has been proved that chrysanthemum extract can improve the specific skin inflammation in mice and has the potential to treat various immune-related skin diseases [8]. Studies have shown that adding 0.5% water extract of chrysanthemum to beauty cream can effectively reduce melanin levels, indicating that chrysanthemum water extract can be used as a natural whitening agent for functional cosmetics [9]. In addition, creams containing chrysanthemums were more effective than placebo in reducing the erythema of rosacea, suggesting that chrysanthemums and their nonmedicinal parts with similar activity have a broad application prospect in cosmetics [10,11].
Remarkable differences existed in the active components of chrysanthemum at different flowering stages and in different parts, and the active functions were related to them to some extent [12]. Comparing the chemical constituents and antioxidant activities of Chrysanthemum (Chrysanthemum boreale Makino) essential oils at different flowering stages, it was found that the antioxidant activity was the highest at full flowering stage, followed by vegetative stage and preflowering stage, which was related to the change of eugenol [13]. Previous studies have been conducted on the chemical composition and antimicrobial activity of Compositae plants, such as Chrysanthemum indicum L., Chrysanthemum trifurcatum, Chrysanthemum boreale, and Chrysanthemum coronarium [14,15]. However, no systematic comparative study was made on the constituents of volatile oil in different flowering stages and different parts of Chrysanthemum morifolium Ramat. Most of the researches on the bacteriostatic effect of chrysanthemum essential oil focus on the fields of bacteriostatic agent, disinfectant and substitute for antibiotics, and so on, and there are few researches on bacteria in the fields of daily chemical [16].
In the present study, not only the composition analysis and antibacterial evaluation of C. morifolium essential oil from different flowering stages and different medicinal parts were carried out, but also evaluated for alleviating inflammation of THP-1 cells caused by P. acnes. Moreover, in order to develop an anti-inflammatory and acne-removing skin care product and expand the application of C. morifolium essential oil in daily chemical products, this paper selected Propionibacterium acnes and Staphylococcus aureus, which are prone to occur on the face and cause acne, to carry out anti-inflammatory and bacteriostasis experiments, providing a certain support for the preparation of anti-inflammatory and acne-removing agents.

Essential Oils
Extraction. JM, TJ, JH, SLC, and RC of the same weight (200 g each) were weighed and placed in the volatile oil extractor, respectively, and extracted by hydrodistillation extraction (HDE) method for 8 h. e essential oils obtained were dried with anhydrous sodium sulphate and stored in the dark at −30°C for subsequent experiments. Yields based on dry weight of the samples were calculated.

GC-MS Analyses.
e essential oils were analyzed using a Clarus 680 GC-MS system (Perkin Elmer, USA) equipped with a 30 m × 0.25 mm × 0.25 μm Elite-5MS column (5% diphenyl 1: 95% dimethylpolysiloxane). Essential oils' components were qualitatively and quantitatively determined using high-resolution gas chromatography/quadrupole tandem time-of-flight mass spectrometry (AxION ® iQT ™ GC/MS/MS). Because it is equipped with a cold EI source, it can effectively generate molecular ion peaks, improving the accurate characterization of compounds. e heating procedure was to keep the initial temperature at 70°C for 2 min, increasing to 130°C at a rate of 10°C/min; then programmed temperature was raised to 150°C at a rate of 1°C/min, increasing to 210°C at 4°C/min and finally held isothermal for 3 min. e solvent delay time was 4 min. e carrier gas was helium at a flow rate of 1.0 mL/min; split ratio was 10 : 1. Injector temperature was 260°C; diluted samples (1.0 μl, 1/100 in hexane) were injected manually. MS parameters were as follows: EI mode, ion source temperature: 180°C; and scan range (m/z): 50-620.

Identification and Quantification of the Essential Oils'
Components. Mass spectrometry and chromatogram analysis were performed using AxION eCipher software, which is compatible with Perkin Elmer Clarus 680 GC-MS. e components were preliminarily matched by comparison of their mass spectra with those of NIST 2.0 library data of the GC-MS system. e relative retention index was calculated based on the same series of n-alkanes (C7-C40), and the volatile components in essential oils were further confirmed by combining with published relevant literature. Evidence-Based Complementary and Alternative Medicine and BHI (Brain-Heart Infusion) broth, and adjusted to approximately 10 8 cfu/mL, respectively.

Microdilution
Method. e antibacterial activity of six essential oils was evaluated by broth microdilution method. Anhydrous ethanol was used as solvent to prepare 1000 mg/ mL mother liquor of essential oil; all tests were performed in MH broth and BHI broth. S. aureus was incubated overnight in a constant temperature chamber (12 h, 37°C, 5% CO 2 ), while P. acnes was cultured in an anaerobic chamber (48 h, 37°C, 10% CO 2 , 10% H 2 , 80% N 2 ), and the final concentration of each strain was adjusted to 3 × 10 4 CFU/mL. Serial doubling dilutions of the oils were prepared in a 96-well microtiter plate with concentrations ranging from 0.195 to 100.00 mg/mL, and then 100 μL bacterial solution was successively added and incubated in an incubator for 12 h or 24 h to observe whether the bacterial solution was turbidized. If there was no turbidized precipitation, the growth activity of bacteria was inhibited; the concentration of essential oil was MIC at this point. MIC was defined as the minimum inhibitory concentration of essential oil against bacteria at which microorganisms do not demonstrate visible growth [17]. Ethanol used for solubilizing the oil was used as solvent control, and the blank control was used without any added drug.
Control was performed for every assay and showed no inhibition of the microbial growth, except blank control. Penicillin and erythromycin lactobionate were used as positive antibiotics for S. aureus and P. acnes, respectively [8]. All inhibition measurements were repeated in triplicate.

Effects of JH and SLC Essential Oils on THP-1 Inflammation
2.6.1. Cell Culture of THP-1 Cells. A human monocytic cell line THP-1 cell was obtained from the Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., and grown in RPMI-1640 medium accompanied with 10% fetal bovine serum, 1% antibiotics, and β-mercaptoethanol 50 μmol/L. e cells were maintained under the environment of 37°C temperature, 5% CO 2 , and 95% of air.

Cytotoxicity Assay.
e CCK8 assay was used to assess the THP-1 cell viability and cell proliferation under the action of JH and SLC essential oils. Cells at exponential growth stage were inoculated in 96-well culture plates and the cell density was adjusted to about 9 × 10 4 cells/mL by RPMI-1640 medium. JH and SLC essential oils with different mass concentrations (final mass concentrations 6.25, 12.5, 25, 50, and 100 mg/mL) were added and incubated at 37°C for 36 h. After centrifugation for 5 min at 800 rmp, the medium was discarded and 100 μL 10% CCK8 solution was added. Absorbance values were measured at 450 nm using a 96-well microplate reader after further incubation for 3.5 h. All experiments were performed in triplicate, and cell viability was expressed as percent change relative to vehicleuntreated control cells.
2.6.3. P. acnes Culture. P. acnes (ATCC6919) was cultured to logarithmic growth stage and its OD600 was adjusted to 1.0. e logarithmic bacterial culture was centrifuged at 4000 rpm at 4°C for 15 min, and the culture supernatant was discarded. e bacterial precipitation was placed in 100 mL phosphate buffer salt (PBS), washed three times, and finally suspended in 10 mL PBS. e bacterial PBS suspension was placed at 80°C for 30 min to achieve heat inactivation reaction. e thermally inactivated P. acnes suspension was stored at 4°C until use.

ELISA for Cytokine
Measurements. THP-1 cells were inoculated into 96-well plates containing complete culture medium, and the concentration of cells was adjusted to 1 × 10 5 cells/mL. After 24 h of culture, fresh culture medium containing JH and SLC essential oil (50 mg/L) was added again for 4 h, respectively. e cells were treated with heatkilled P. acnes co-cultured for 8 h. Centrifuged at 3000 rpm/ min for 20 min, the supernatant was carefully collected into the ribozyme-free EP. e level of IL-1β in cell supernatant was determined by ELISA kit [18].

Percentage Extractive Yield of Essential Oils.
By hydrodistillation of air-dried JM, TJ, JH, SLC, and RC, the yields of the six essential oils obtained are 0.44%, 0.35%, 0.21%, 0.16%, and 0.12% (all the above proportions are the quality of essential oil/weight of dried medicinal materials), respectively, with the highest yield for the JM oil (0.44%) and the lowest yield for the RC oil (0.025%) among the five different states of the medicinal plant Chrysanthemum morifolium. e afforded JM, TJ, JH, and SLC essential oils have light yellow and pleasant aroma at room temperature, while the essential oils obtained by RC distillation are orange-red and have a pleasant woody fragrance.

Chemical Composition of the Essential Oils.
Qualitative and quantitative analytical results by GC and GC/MS are shown in Table 1. And the typical total ion current (TIC) of six essential oil samples of C. morifolium is shown in Figure 1. e identification of the oil components was based on the comparison of their mass spectra and retention indices (RI) with those reported in the literature [19][20][21][22][23]. e chromatographic analysis resulted in the obtainment of 124 components and identification of 114 components, representing 89.25%∼96.70% of the six essential oils. Table 1, compared with JM, TJ, and JH from three different flowering stages, the contents of heterospiroolefins, sesquiterpene hydrocarbons, and hydrocarbon in the volatile oil decreased gradually with the growth of flowering stage. Among them, the contents of heterospiroolefins decreased significantly from 44.81% in JM essential oil to7.27% in JH. Furthermore, the average Evidence-Based Complementary and Alternative Medicine  Additionally, the highest content of sesquiterpenoids in TJ (6.44%) and JH (28.62%) was α-cadinol, while the highest content of sesquiterpenoids in JM is β-sesquiphellandrene, with a content of 2.21%. Sesquiterpenoids play an extremely important role in living organisms and have a variety of pharmacological effects, which laid a certain foundation for the development and application of chrysanthemum essential oil in cosmetics and medicine.

Comparison of Chrysanthemum Essential Oils at
Different Parts. Remarkable differences were also observed between the flowerhead, roots, leaves, and stems of C. morifolium. In terms of the number of compounds identified, the number of compounds identified from JH, SLC, and RC was 77, 71, and 57, respectively. e number of JH compounds is much higher than that of RC compounds.  JH (96.70%), SLC (94.28), and RC (89.25%) also had certain differences for the relative content of essential oils obtained. e content of sesquiterpenes in SLC (27.76%) was 5.7 and 64 times that of JH and RC, respectively. e major compound of sesquiterpenes in SLC was caryophyllene (8.84%), while the main compound of sesquiterpenes in JH and RC was iso-caryophyllene (1.77%) and (E)-β-farnesene (0.11%), respectively. Natural bicyclic sesquiterpenes, caryophyllene are present in a large number of plants worldwide. Caryophyllene possesses significant anticancer and analgesic activity, and it can improve the systemic inflammation and oxidative status of arthritic rats [24]. In addition, caryophyllene is also approved as food additive, taste enhancer, and flavoring agent and termed as a phytocannabinoid. Present results indicate that the most plentiful constituent of flowerheads essential oils from C. morifolium was α-cadinol (28.62%). But in the SLC and RC oils, (E)-tibetin spiroether (20.53%) and 2-trans, 6-trans-farnesyl acetate (32.00%) were the predominant compound, respectively. However, the essential oils from all parts of C. morifolium were rich in terpenoid components, which is consistent with the research results of most researchers. e results clearly revealed that the JM, TJ, and SLC oils were dominated by heterospiroolefins with the major compound being (E)-tibetin spiroether, but JH oil was dominated by oxygenated sesquiterpenes with the major constituent was α-cadinol. (E)-Tibetin spiroether was first reported in Artemisia roxburghiana Besser essential oil [25] but never appeared in Chrysanthemum morifolium essential oil. Due to its high proportion in JM, TJ, and SLC essential oils, the chemical composition and pharmacological action of (E)-tibetin spiroether are worth our attention.

Antimicrobial Activity.
e antibacterial activity of C. morifolium essential oils against 2 types of Gram-positive bacteria (S. aureus and P. acnes) was evaluated by the microdilution method to determinate the minimum inhibitory concentration (MIC) ( Table 2).
To varying extents, the six essential oils (JM, TJ, JH, SLC, and RC) inhibited the growth of two bacteria commonly found in cosmetics according to the results given in Table 2.
e minimum inhibitory concentrations (MIC) of JM, TJ, and JH volatile oils against S. aureus and P. acnes were 10 mg/mL and 25 mg/mL, respectively. By comparing the oils from different parts, the JH oil was found to be the most effective, followed by SLC and RC for S. aureus, on which JH oil shows twice and five times inhibitory effect that of SLC and RC oil. e content of terpenoids in JH essential oil reached 78.74%, which was much higher than SLC (40.13%) and RC (41.82%) oils. e above results are consistent with literature reports, indicating that terpenoids in essential oils have significant antimicrobial activity, and terpenoids can cause bacterial death by destroying the integrity of cell membranes or making bacteria lose part of their functions [26]. For SLC essential oil, its inhibitory effect on S. aureus was lower than that of JH, and its MIC value was 20 mg/mL. e roots' oil showed the lowest activity against all strains, and its MIC value was 50 mg/mL. is may be due to the fact that the main metabolites of the esters fraction in RC volatile oil were 2-trans, 6-trans farnesyl acetate. e main components of other five oils are (E)-tibetin spiroether, except the JH oil with α-cadinol containing oxygen sesquiterpenes. Since these five essential oils have similar antibacterial activities against these two bacteria, we can speculate that α-cadinol and (E)-tibetin spiroether may have some similar antibacterial activities.
To the best of our knowledge, this is the first time that chrysanthemum essential oil has been studied against P. acnes is a common, chronic skin disease that arises in the hair follicle and often involves inflammation [27]. It has been reported that P. acnes is thought to trigger inflammatory responses and lead to subclinical and inflammatory acne lesions [28]. In the treatment of acne, erythromycin, clindamycin, and other antibiotic preparations are often used, easy to cause bacterial resistance [29], so it is necessary to study the inhibitory effect of natural plants on the growth of P. acnes. To determine the cytotoxicity, THP-1 cells treated with different concentrations of JH and SLC essential oils (100, 50, 25, 12.5, and 6.25 mg/L) for 36 h were analyzed by an established CCK-8 assay. e effects of JH and SLC essential oils at different concentrations on the survival rate of THP-1 are shown in Figure 2. JH and SLC essential oils have no toxic effects on THP-1 cells within the range of the measured mass concentration (6.26 mg/L-100 mg/L), which fully demonstrates the safety of JH and SLC essential oils. It can be seen from Figure 2 that when the mass concentration of JH and SLC essential oil is 50 mg/L, it has a certain effect on the proliferation of THP-1 cells, and the cell morphology at this time has no obvious change from that before the treatment, and it is still bright and full. erefore, the optimal concentration of JH and SLC essential oils on THP-1 was determined to be 50 mg/L, and subsequent efficacy screening tests were conducted with this mass concentration.

Effects of JH and SLC Essential Oils on Proinflammatory Cytokines in Heat-Killed P. acnes-Treated THP-1
Cells. We first examined how much volume (10 μL, 30 μL, and 50 μL) of heat-killed P. acnes suspension was capable of inducing secreted inflammatory cytokines. e expression level of proinflammatory cytokines was measured by ELISA. As can be seen from Figure 3(a), when the liquid volume of P. acnes inactivated bacteria was 30 μL, the inflammatory cytokines released were significantly different from those of the blank group, so 30 μL P. acnes suspension was selected for the next experiment. Heat-killed P. acnes increased the secretion of IL-1β compared with the normal control (KB) in THP-1 cells. However, JH and SLC essential oils treatment significantly inhibited the secretion of cytokine. us, these observations suggest that JH and SLC essential oils effectively inhibit the secretion of IL-1β in THP-1 cells (Figure 3(b)). In addition, JH essential oil has a better effect than SLC essential oil in alleviating inflammation of THP-1 cells, which may be related to the high content of sesquiterpenes in JH essential oil [30,31].

Conclusions
In this paper, a total of 124 compounds were obtained and 114 compounds were identified from 6 volatile oils sample of C. morifolium. e main constituents were terpenoids, heterospiroolefins, and esters compounds, including alkanes, fatty acids, phenols, alcohols, and aldehydes. Among them, the content of terpenoids in the essential oil of . e data are presented as the mean ± SD. * p < 0.01, * * p < 0.01, and * * * p < 0.001 versus the control group, by one-way ANOVA with Tukey's test.